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Modeling Plasmid Selection. And Authentic Assessment. 6-2010. http://biology2020.wikispaces.com/. With the pGlo Plasmid. Bacteria are very useful organisms in genetic engineering. They are able to bring in plasmids. Into which DNA of interest may be added. The Problem?. - PowerPoint PPT Presentation
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6-2010
Modeling Plasmid Selection
And Authentic Assessment
http://biology2020.wikispaces.com/
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With the pGlo Plasmid
Bacteria are very useful organisms in genetic engineering.
They are able to bring in plasmids.
Into which DNA of interest may be added.
The Problem?
• Finding the bacteria that have taken in the modified plasmids.
• You can see the bacteria, but not the plasmid.
•Must look for some visible effect of the plasmid.
What to look for?
•Growing in the presence of antibiotic.
• The effect of a gene, like the glow of pGLO
Cut Four Paper Ovals(or…draw 4 ovals on a large sheet of
paper.)
• Each represents a bacterial cell
Make Four Chromosomes
•Out of four chenille stems• Bacterial• Circular•Give a little twist to prevent confusion with a nucleus.• Add to “cells.”• (Or just draw this chromosome in on paper)
Form your plasmidsUse 1/2 a chenille stem each. Make two plasmids as below.
bla Ara C
Antibiotic Resistance
Ara B Ara A AraD
GFP
Reserve
Twist into ring but leave tails
Bla codes for beta lactamase, a protein which confers
antibiotic resistance.
Structural Genes
Regulatory Gene
By the way…great place to teach the operon.
Restriction Enzymes
• Plasmids are cut with the same restriction enzyme used to cut the DNA to be inserted. A restriction enzyme which leaves overhanging sticky ends is needed for this this procedure. This provides the free base pairs needed to combine the plasmid DNA with the source DNA.
Restriction Enzyme Cut from EcoRI
✤Procedure:•
• Open the plasmids you prepared by untwisting the ½ chenille stem.
• This represents the recognition site for the restriction enzyme. Remove ara B, araA, and araD (last 4 beads).
• Add a green bead to represent the GFP (gene of interest).
• GFP shares the same sticky ends as the plasmid.
Modeling pGlo
-pGLO -pGLO +pGLO +pGLO
LB LB/AMP LB/AMP LB/AMP/ARA
Bacteria Lives?
-pGLO -pGLO +pGLO +pGLO
LB LB/AMP LB/AMP LB/AMP/ARA
Yes No Yes Yes
Bacteria Glows?
-pGLO -pGLO +pGLO +pGLO
LB LB/AMP LB/AMP LB/AMP/ARA
No No No Yes
Great Lab for Authentic Assessment
Let’s try it.
More Authentic Assessment
1. Is this gel in the chamber correctly?
2. How do you know?
2nd block may get this!
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Provide a ruler and some semi-log paper.
Ask: How big is fragment X?
Which is larger , X or Y?
Micropipettor• Have out a micropipettor
and a microtube.
• Instructions:– Pipet 50 µL of water into the
microtube.
– Close and write your initials on the top with the permanent marker.
– Place in the box by clicker number.
– Set the micropipettor to the day of the month you were born on.
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Osmosis• Provide a scale and weigh
boat. Just use water…but ask…
• This bag has been in this beaker of distilled water for 30 minutes.
• The initial mass of the bag was
30 g.
• Is the bag hypertonic, hypotonic, or isotonic to the beaker?
• Which way did water move?19
Fruit Flies
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Give sex and eye type.
Mitosis
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Identify the stage
of mitosis
at the tip of the
pointer.
Respiration
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Read the pipette.
References
• Biotechnology Explorer, pGLO Bacterial Transformation Kit. BioRad.
http://www3.bio-rad.com/images/pglomap2.gif
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